Cellular mediators of innate immunity have the capacity to recognize and respond to target cells in the absence of antibody or T cells. Recently an ancient family of pathogen recognition receptors, the toll like receptors (TLRs) was discovered in humans and mice and several have been shown to trigger inflammatory responses to pathogen derived substances. We and others have shown that immature dendritic cells (iDC) express TLRs, and that TLRs on iDC drive maturation in response to pathogens, an essential component of acquired immunity. The TLR-driven maturation is modulated by G-protein coupled receptors, and we have also shown that the H2 histamine receptor, in particular, can profoundly affect the maturation process. Histamine is produced by mast cells, which are located in the same microenvironment as DC, and we demonstrated that human monocyte-derived iDC express two active histamine receptors, H1 and H2. Although histamine failed to affect the LPS-driven maturation of iDC with regard to phenotypic changes or capacity to prime naive T cells, it dramatically altered the repertoire of cytokines and chemokines secreted by mature DC. In monocyte-derived DC histamine increased IL-10 and reduced IL-12 secretion, and in plasmacytoid DC histamine caused a decrease in type-I interferon production. As a result of its affects upon cytokine secretion, histamine-treated DC induced a Th2 phenotype during T cell priming. We are currently testing in mice the hypothesis that maturation of DC in a microenvironment that is rich in histamine will promote the generation of Th2 cells in the draining lymph nodes. To do this we have established a system in which T cells from TCR transgenic mice are adoptively transferred into syngeneic normal mice. The TCR is specific for ovalbumin, and we have shown that subcutaneous injection with the antigen together with either LPS or CpG ODNs (which stimulate TLR4 and TLR9 respectively) as adjuvant induce proliferation of nave transgenic T cells in the draining lymph nodes. The object of this study, currently being tested, is to see if stimulators of mast cell degranulation, administered with antigen and adjuvant can modify the polarization of T cells in the lymph nodes. The TLR4 response to LPS requires the binding of MD-2 to its extracellular domain. MD-2 contains a leader sequence but lacks a transmembrane domain, and we asked whether it is secreted into the medium as an active protein. As a source of secreted MD-2 (sMD-2) we used culture supernatants from cells stably transduced with epitope-tagged human MD-2. We showed that sMD-2 exists as a heterogeneous collection of large disulfide linked oligomers formed from stable dimeric subunits, and that concentrations of sMD-2 as low as 50 pM enhanced the responsiveness of TLR4 reporter cells to LPS. An MD-2 like activity was also released by monocyte-derived iDC from normal donors. When co-expressed with MD-2, TLR4 indiscriminantly associated in the ER/cis Golgi with different sized oligomers of MD-2, and excess MD-2 was secreted into the medium. We concluded that normal and transfected cells secrete a soluble form of MD-2 that binds with high affinity to TLR4. Thus, sMD-2 might play a role in regulating responses to LPS and other pathogen derived substances in vivo. We have shown that secreted MD-2 binds to LPS and to TLR4 on cell surfaces. At 37 C the MD-2 rapidly loses its capacity to bind to both LPS and TLR4, but that LPS stabilizes MD-2. The stabilization requires LPS to be incubated with MD-2 for several hr at 37 C, in the presence of CD14, a molecule know to be important in transferring LPS to the TLR4 MD-2 complex. We are currently testing whether LPS first binds to MD-2, and whether the LPS-MD-2 complex is sufficient to trigger TLR4 activation. TLR9 recognizes bacterial DNA and oligodeoxynucleotides (ODN) containing unmethylated CpG motifs. TLR9 is not located on the cell surface, but rather interacts with bacterial DNA in an interior compartment., as expected, since DNA is released from bacteria after phagocytosis. We have shown that both the cytoplasmic and ecto-domains of TLR9 contain localization signals, and have located one of these signals in the TLR9 cytoplasmic domain by mutational analysis. Interestingly, TLR9 appears to be located, both in normal and transfected cells, in the endoplasmic reticulum, as indicated by confocal microscopic analyses, and sensitivity to endo-H digestion. We are currently defining a second localization signal in the cytoplasmic domain of TLR9, and are initiating studies aimed at locating the internalization signal in the TLR9 extracellular domain.